Project Summary Thousands of proteins in the human proteome are subjected to modification by reversible lysine acetylation, which is now recognized as a key regulator of diverse processes such as epigenetics and metabolism. The careful balance between the expression and activity of lysine acetyl transferases (KATs) and lysine deacetylases (KDACs) maintains the acetylome of a cell. Our long-term goal is to develop a mechanistic understanding of how lysine acetylation is controlled and how the mark effects cell state. Our current focus is to develop a new class of small molecule fluorescent chemical tools that report on lysine deacetylation activities in living cells with spatial resolution. We will deploy this new family of chemical tools to test the hypothesis that KDAC signaling is in part mediated by subcellular distribution, which controls access to substrates and local cofactors. This hypothesis could help explain some of the ambiguous results associated with lysine acetylation that have been observed, as the biological consequence of modulation of a specific KDAC isoform may be masked by other isoforms in a cell-type or disease specific manner. We postulate that a key to understanding how lysine acetylation is regulated is to monitor the overall amounts of KAT and KDAC activities with spatial-temporal resolution. Our primary biological interests right now deal with roles of KDACs outside of the nucleus, in particular in the mitochondria and the cytoplasm, while pursing mechanistic studies in the context of metabolism and breast cancer.